Pest control and particularly insect and/or arachnid control, especially control of household insects, ecto-parasites and insects relevant for public health and hygiene (e.g. urban protection) such as cockroaches, fleas, ants, termites, earwigs, mosquitos, flies and house crickets is an important field. The presence of insects in locations such as at home, in offices, restaurants, hospitals or warehouses undoubtedly causes distress because there is a common public perception that insects such as cockroaches or flies live in places that are dirty and not well kept.
These insects do not only causes distress but also contaminate food and eating utensils, destroy fabric and paper products and impart stains and unpleasant odours to surfaces they contact. Furthermore, these insects can pose health risks as carriers for bacteria. For example, cockroaches may transmit bacteria that cause food poisoning (Salmonella spp. and Shigella spp.). German cockroaches are believed capable of transmitting disease-causing organisms such as Staphylococcus spp., Streptococcus spp., hepatitis virus and coliform bacteria. They also have been implicated in the spread of typhoid and dysentery. Some people, especially those with asthma, are sensitive to the allergens produced by these cockroaches.
There are various chemical insecticides and capturing devices developed and commercially available for fighting household pests. However, increasing efficacy of these means is usually linked with increased health risk. Insecticides may contaminate food which is nearly unavoidable in places such as kitchens, restaurants or food storages and incorporation may cause health risks to humans.
The solution to this problem of contamination has been to use less toxic insecticides. However, when applying less toxic insecticides, there is an increased probability that the insect may become resistant over time. Insecticides act by binding to a certain insect protein, such as an acetylcholine receptor for example, and cause death of the pest species by either deactivating or over-activating the protein. Insecticides have been developed to be safe at certain concentration, but can and do impact on human health when incorporated at higher dosages or over long periods. In contrary to agrochemicals, household insecticides are applied in places where food is stored or prepared and food contamination and contact to humans cannot be avoided.
One alternative to chemical pesticides is to utilise biological agents. Over the last few years, downregulation of genes (also referred to as “gene silencing”) in multicellular organisms by means of RNA interference or “RNAi” has become a well-established technique.
In general, RNAi comprises contacting the organism with a double stranded RNA fragment or “dsRNA” (generally either as two annealed complementary single strands of RNA or as a hairpin construct) that comprises a nucleotide sequence that corresponds to (at least part of) the nucleotide sequence of the gene to be downregulated (the “target gene”). Reference is inter alia made to the International application WO 99/32619 (Carnegie Institute of Washington), the International application WO 99/53050 (CSIRO), the International application WO 00/01846 (Devgen) and to Fire et al., Nature, Vol. 391, pp. 806-811, February 1998.
In nematodes, RNAi can be performed by feeding the nematode with the dsRNA fragment as such, or alternatively with a bacterial strain that either contains the dsRNA fragment or that upon ingestion by the nematode is capable of expressing the dsRNA fragment. For this so-called “RNAi by feeding”, reference is inter alia made to the International application WO 00/01846 by applicant, and to WO 99/32619 cited above, in which the nematode C. elegans is used.
Many dsRNA constructs have been described in the art. A classic dsRNA is produced from a DNA construct comprising two convergent promoters flanking the sequence complementary to the target sequence which needs to be downregulated (see for example WO00/01846 (Devgen)). As the technology of dsRNA mediated gene silencing advanced, new constructs were designed to improve the dsRNA for various purposes.
In order to produce the dsRNA more efficiently, a stem-loop-stem structure or “hairpin” was developed. As described in, for example, document WO 99/53050 (CSIRO), this hairpin allows the formation of dsRNA from one single RNA transcript. The RNA transcript comprises the sense and anti-sense version of the complementary sequence, separated by a non-complementary loop structure allowing the RNA transcript to fold back and to base pair into a dsRNA stem portion.
DsRNA gene silencing finds application in many different areas, such as for example dsRNA mediated gene silencing in clinical applications (WO2004/001013) and in plants. In plants, dsRNA constructs useful for gene silencing have also been designed to be cleaved and to be processed into Short interfering RNAs (siRNAs).
RNAi has also been proposed as a means of protecting plants against plant parasitic nematodes, i.e. by expressing in the plant (e.g. in the entire plant, or in a part, tissue or cell of a plant) one or more nucleotide sequences that form a dsRNA fragment that corresponds to a target gene in the plant parasitic nematode that is essential for its growth, reproduction and/or survival. Reference may be made to the International application WO 00/01846 by the present applicant, U.S. Pat. No. 6,506,559 (based on WO 99/32619), and to International applications WO 01/96584, WO 01/37654 and WO 03/052110 for a description of such techniques.
Elbashir et al. (Nature, 411, 494-498, 2001) have demonstrated effective RNAi-mediated gene silencing in mammalian cells using dsRNA fragments of 21 nucleotides in length (also termed small interfering RNAs or siRNAs).
WO 03/004644 describes delivery of dsRNA to arthropods in general terms and is incorporated herein by reference. WO 03/004644 details down regulation of the reporter gene GUS (Clonetech) using RNAi in Drosophila melanogaster and down regulation of the vATPase gene in H. armigera. 
WO 01/34815 relates to baculovirus expression vectors which produce dsRNA and the use of these vectors in pest control.
Although the technique of RNAi has been generally known in the art in plants, nematodes and mammalian cells for some years, to date little is known about the use of RNAi to down-regulate gene expression in insects and/or arachnids. In addition, little is known on the application of RNAi to control pest species such as household insects, ecto-parasites and insects and/or arachnids relevant for public health and hygiene.
Constructs suitable and efficient for dsRNA mediated pest control, should meet at least some of the following requirements                (1) the dsRNA must be taken up by the pest organisms        (2) the dsRNA must have good stability in the pest organisms        (3) the dsRNA must be effective in the pest organism to control its viability, growth and/or development and/or        (4) the dsRNA must guarantee maximized safety and minimized environmental impact.        
It is now the purpose of the present invention to provide dsRNA constructs, which meet the above-mentioned requirements.